Nutrient excess in a stabilized co-culture system of Caco2/HT-29 cells: an ultrastructural and functional time-course study

The intestine represents one of the most important barriers of our body and interacts with several exogenous substances, among which nutrients. Today, the effects due to an excess of nutrients on intestinal morpho-functional changes, similar to the ones found in obesity, have been studied only in in vivo animal models. Many experimental difficulties hampered in establishing a physiological long-term experimental model starting from primary cultures of normal small intestinal and colon cells. For this reason, an intestinal Caco2/HT-29 (70/30) co-culture was set up in our lab starting from the differentiated parental cell populations to mimic the human intestinal epithelium. Co-culture was harvested at confluence (T0) and at 3, 7, and 15 days (T3, T7, and T15, respectively) post-confluence. Ultrastructural (TEM) and functional analysis (Alkaline Phosphatase, ALP; Aminopeptidase N, APN; Dipeptidyl Peptidase-IV , DPPIV; Transepithelial Electrical Resistance, TEER) were carried out. In the present study, two parallel experimental groups were cultured: the standard group and the excess group. In the standard group, the culture medium was changed every four days, whilst in the excess group on alternate days from T0. Transmission electron microscopy revealed that the excess of nutrients drives co-cultures towards a less differentiated absorptive phenotype. On the other hand, mucus granule presence was more and more evident from T3. The specific activity of ALP and APN, known markers of intestinal differentiation, and that of DPPIV, a specific marker of enterocyte differentiation, progressively increased. TEER, indicative of the barrier properties of the co-culture, increased at post confluence up to T15. In conclusion, data here presented show that the excess of nutrients can directly modify both morphology and function of the intestinal cells, opening the way to study at the effects due to specific nutrients on cell proliferation and differentiation involved in the acquisition of an obese human phenotype

Morphological analysis of JAK1 intracellular pathway activation after pro-inflammatory psoriatic cytokines exposure: inside-out and outside-in the epidermis

For their normal growth, cells depend on a continuous flow of signals from the environment. The Janus kinases (JAK) 1 transducers signalling pathway is a pleiotropic cascade used to transduce a multitude of signals among cells. A variety of ligands including cytokines, hormones, growth factors, and their receptors stimulate the JAK1 pathway. Cytokines, a large and very heterogeneous family of small and generally soluble glycoproteins, both control multiple biological processes as haematopoiesis, inflammation, and immunity playing a central role in cell-cell communication. Their action is mediated by the binding to specific receptors on the cell surface, thus transducing biological information to target cells [1]. Pro-inflammatory cytokines play a pivotal role in several inflammatory illnesses including psoriasis. Among them, interleukin (IL)-17, IL-22, IL-23 and tumor necrosis factor (TNF)-alpha play a central role. In the formation and progression of the psoriatic lesion a typical marker is keratin (K) 17 which is correlated with psoriasis severity. The aims of this study were to evaluate the early, direct, and specific effects of pro-inflammatory psoriatic cytokines i) on the activation of the intracellular pathway JAK1 and ii) on the correlation with the induction of K17 expression in a three-dimensional model (3D) of human skin (n=7) by immunofluorescence. Biopsies were cultured overnight epidermal side-up in a Transwell system and exposed to 50 ng/ml IL-17, or 100 ng/ml IL-22, or 50 ng/ml IL-23 or 100 ng/ml TNF-alpha. Samples were harvested 24 (T24), 48 (T48), and 72 (T72) hours after cytokine incubation. In samples not exposed to cytokines, a JAK1 slight labelling was observed throughout the epidermis, decreasing at T72 in the lower layers. At T24, IL-17 and IL-22, but not IL-23 and TNF-alpha, induced an expression of JAK1 in the spinous layer. At T72, JAK1 immunostaining decreased in all samples, similarly to controls. K17 immunopositivity was induced and progressively increased with time in the suprabasal layers of epidermis in all experimental groups, with the exception of the TNF-alpha group. These results suggest that cytokines exert parallel effects on JAK1 pathway activation and K17 induction. In conclusion, 3D this model, reproducing some features of psoriatic microenvironment, represents a useful experimental approach to dissect the specific role of each cytokine in the different steps of psoriatic lesion formation

The psoriatic shift induced by interleukin 17 is promptly reverted by a specific anti-IL-17A agent in a three-dimensional organotypic model of normal human skin culture

Interleukin 17A (IL-17A), mainly produced by the T helper subclass Th17, plays a key role in the psoriatic plaque formation and progression. The clinical effectiveness of anti-IL-17A agents is documented, but the early and specific mechanisms of their protection are not identified yet. The challenge of the present study is to investigate the possible reversal exerted by a specific anti-IL-17A agent on the psoriatic events induced by IL-17A in a three-dimensional organotypic model of normal human skin. Bioptic skin fragments obtained after aesthetic surgery of healthy women (n=5) were incubated with i) IL-17A biological inhibitor (anti-IL-17A), ii) IL-17A, iii) a combination of IL-17A and its specific IL-17A biological inhibitor (COMBO). A Control group was in parallel cultured and incubation lasted for 24 and 48 h epidermal-side-up at the air-liquid interface. All subjects were represented in all experimental groups at all considered time-points. Keratinocyte proliferation and the presence of epidermal Langerhans cells were quantitatively estimated. In parallel with transmission electron microscopy analysis, immunofluorescence studies for the epidermal distribution of keratin (K)10, K14, K16, K17, filaggrin/occludin, Toll-like Receptor 4, and Nuclear Factor kB were performed. IL-17A inhibited cell proliferation and induced K17 expression, while samples incubated with the anti-IL-17A agent were comparable to controls. In the COMBO group the IL-17A-induced effects were almost completely reverted. Our study, for the first time, elucidates the most specific psoriatic cellular events that can be partially affected or completely reverted by a specific anti-IL-17A agent during the early phases of the plaque onset and progression. On the whole, this work contributes to expand the knowledge of the psoriatic tableau

Modulation of epidermal proliferation and terminal differentiation in a promising ex vivo human skin model mimicking a psoriatic microenvironment

Epidermal keratinocyte hyperproliferation is one of the key features involved in the formation/progression of psoriatic lesions and is driven by cytokines, among which TNF-\u3b1, IL-17, IL-22 and IL-23, secreted by both activated resident immune cells and keratinocytes (1). The network orchestrated by these cytokines is essential for the communication between resident cells and infiltrating cells and is due to redundancy, synergism and, sometimes, the reciprocal antagonism of cytokines. The aims of our study were to investigate whether the exposure of normal human skin to four main psoriatic cytokines, i.e. TNF-\u3b1, IL-17, IL-22, and IL-23 (cytokine mix) induced i) a modulation of epidermal proliferation and ii) a modification of keratinocyte terminal differentiation (TD). Human skin samples (n = 5) obtained from healthy 20-40 years-old women after plastic surgery, were exposed to the cytokine mix in a Transwell system at air-liquid interface as previously described (2). For each patient a control (ctr) group was not exposed to cytokine mix. Samples were harvested 5 (T5), 24 (T24), 48 (T48) and 72 (T72) hours after cytokine stimulation, processed for paraffin embedding and immunofluorescence analysis for the quantitative analysis of epidermal proliferation and the expression of the TD biomarkers, keratin (K) 10 and 17. A decrease of cell proliferation was evident starting from T5 in samples exposed to cytokine mix and was progressively more marked at later time points (T5 ctr 47.44 \ub1 6.90 vs mix 23.07 \ub1 8.84; T24 ctr 41.12 \ub1 12.78 vs mix 12.16 \ub1 1.53; T48 ctr 25.88 \ub1 10.21 vs mix 2.19 \ub1 2.44; T72 ctr 10.49 \ub1 2.52 vs mix 0.65 \ub1 1.02) (p<0.05). K17 expression was evident in samples exposed to the cytokine mix. Altogether the present results suggest that cell proliferation inhibition and K17 expression could be regarded as the basis for a later response to injury leading to psoriatic lesion formation/progression. In conclusion, this model allows to investigate the intimate interplay among different psoriatic cytokines and new insights may be of potential value for future clinical treatments

Effects of UV rays and natural compound repairs using an ex-vivo human skin model: morphological and genotoxicological analysis

Among the key factors in skin disorders such as wrinkling, dryness and photo-aging, the exposure to solar ultraviolet (UV) radiation plays a central role (1). Recently, compounds rich in polyphenols such as Thymus Vulgaris Leaf (TVL) extract and its major component Thymol (T) have been proposed in the prevention of UV-induced skin damages (2). Experiments were carried out in a human ex-vivo skin model, in which biopsies were obtained from aesthetic surgery of healthy 20-40 year-old women (n=6) after written informed consent (3). After 24 h, samples were pre-treated for 1 h with comparable concentrations of two compounds (TVL: 1.82 \ub5g/mL and T: 1 \ub5g/mL) before being irradiated with different UVB doses (0.24 J/cm2 to 0.72 J/cm2) or UVA radiation (8 J/cm2 to 32 J/cm2). Samples were harvested 24 h after irradiation and were processed both for light and transmission electron microscopy.Cell proliferation, Lactate Dehydrogenase assay, alkaline comet test, and histone H2AX phosphorylationwere evaluated. Both UVB and UVA induced an early inhibition of cell proliferation and DNA damage compared with respective controls. In particular, UVB rays were always more cytotoxic and genotoxic than UVA. The T-pretreatment showed a reduction of UVB-induced structural/ultrastructural and genotoxic damages. These results suggest that polyphenol fraction of tested substances may be useful for skin photoprotection after UV radiation damage in an ex-vivo human skin model. The present study suggests that this experimental setting can be a reliable approach for safety evaluation of UV skin exposure

The analysis of the dermal collagen matrix in the absence of &#945;11&#946;1-integrins suggests a potential role for integrins &#945;11&#946;1 in the regulation of skin biomechanics

Integrins \u3b111\u3b21 are major collagen receptors and are thought to play a central role in fibrillar collagen arrangement [1;2], but this has not been demonstrated in vivo. In order to answer this question, here, we analysed the overall organisation of the dermal collagen network fibril diameter in samples of back skin of \u3b111\u3b21-integrin-deficient mice (KO). Dermal collagen organisation was assessed for its complexity and its heterogeneity on paraffin sections after Sirius red staining (4 KO and 4 controls), by quantifying fractal dimension and lacunarity respectively. The results showed that fractal dimension was increased in KO mice (1,40\ub10,06 in \u3b111\u3b21 KO mice vs 1,24\ub10,05 of control mice, p=0,009), whereas Lacunarity was reduced (0,78\ub10,06 in \u3b111\u3b21 KO mice 0,97\ub10,02 of control mice p=0,002), indicating a re-organisation of the dermal collagen network in absence of integrins \u3b111\u3b21. Fibril diameter was studied in images taken at the Transmission Electron Microscope (5 KO and 5 controls). The total number of fibrils examined was 22,212 (for the 5 controls) and 28,446 (for the 5 KO). The analysis showed a proportional increase in smaller fibrils with a proportional decrease in larger fibrils in \u3b111\u3b21 KO mice, being these differences were most evident in fibrils with smallest (120nm) diameter. Chi squared test confirmed statistical significance of these changes (equivalent to p=0,001). Given the fundamental role of dermal collagen in skin stability, these changes in collagen organisation and fibril size also suggest a potential implication of \u3b111\u3b21 integrins in the control of skin biomechanics

Interleukin 17 affects early and late biomarkers of terminal differentiation in a three-dimensional model of normal human skin

Interleukin (IL)-17 expression has been correlated with the pathogenesis of multiple autoimmune diseases, as rheumatoid arthritis, multiple sclerosis, and, more recently, psoriasis (1). During plaque formation, the interplay between immunocytes and keratinocytes is deregulated, resulting in the altered expression of keratin (K) 17, occludin, and filaggrin in psoriatic lesional epidermis (2, 3). The involvement of IL-17 in psoriasis pathogenesis has been identified (4), but the specific and intrinsic effects exerted by this cytokine have not been thoroughly investigated. The aim of the present work was to study by indirect immunofluorescence the expressions of K17, K10, filaggrin, and occludin in a three-dimensional model of normal human skin standardized in our laboratory (5). Human skin samples (n = 5) obtained from healthy 20-40 years-old women after plastic surgery, were exposed to IL-17 in a Transwell system at air-liquid interface as previously described (5). Samples were harvested 24 (T24), 48 (T48), and 72 (T72) hours after IL-17 stimulation and processed for paraffin embedding and immunofluorescence analysis for expressions of K17, K10, filaggrin, and occludin. After IL-17 exposition, K17 immunostaining progressively increased with time in the upper stratum spinosum, while K10 expression resulted homogeneously distributed in the suprabasal layers. In IL-17 treated samples occludin staining became irregular starting from 24 hours. On the other hand, filaggrin distribution was affected in T48 samples, where the immunolabelling was discontinuously punctuate. In conclusion, our results strongly support the use of this experimental setting for investigating the time-dependent early effects induced by IL-17 in normal human skin

Effect of TNF-alpha and IL-17 on TLR expression and Langerhans cells phenotype in a three-dimensional model of normal human skin: a morphological study

Toll-like receptors (TLRs) are essential for innate immunity and contribute to create the skin barrier. Their abnormal stimulation is involved in the development of several dermatological diseases, among which psoriasis. Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role in the pathogenesis of psoriatic plaques and their proinflammatory activity can affect Langerhans cell (LC) phenotype. In a well characterized three-dimensional model of organotypic cultures of normal human skin [1-3] we evaluated the effect of TNF-alpha and IL-17 on the expression of TLR2 and 9 by immunofluorescence, on the ultrastructural morphology of keratinocytes and LCs by transmission electron microscopy (TEM). Human skin explants (n=7) were cultured at the air-liquid interface overnight in a Transwell system and exposed to 50 ng/ml IL-17 or 100 ng/ml TNF-alpha or a combination of both cytokines. Samples were harvested 24 (T24) and 48h (T48) after cytokines incubation. After incubation with IL-17 and IL-17+TNF-alpha, TLR2 immunostaining was not detectable in the basal layer, differently from controls and TNF-alpha-treated samples. Conversely, TLR9 expression was progressively induced in granular keratinocytes in all cytokine-exposed groups. By TEM, enlargements of intercellular spaces were evident especially and, after IL-17 treatment, LCs showed an activated phenotype. At T24 LCs number increased indicating that TNF-alpha and IL-17+TNF-alpha exert a chemoattractant activity, while at T48 only IL-17+TNF-alpha maintained this effect on trapping LCs in epidermis. TNF-alpha and IL-17 differently affect LCs behaviour and TLR expression, with a specific contribution to the inflammatory loop underlying the lesion formation. The simultaneous inhibition of the effect of different cytokines - all with a defined role in the pathogenesis of psoriasis - could further improve psoriasis treatment

Co-culture of Caco2 and HT-29 cells as an innovative method to mimic in vitro the morphology and permeability properties of human intestinal epithelium

For investigating the complexity of the human intestinal epithelium, a valid experimental approach is represented by co-culture. In the present study an intestinal co-culture Caco2/HT-29 (70/30) was set up starting from the parental populations of differentiated cells as previously described [1, 2]. Co-culture was harvested at 0 (T0), 6 (T6), and 14 (T14) days of post confluence after plating. Transmission electron microscopy was carried out to monitor the morphological features of cell differentiation. Alkaline Phosphatase (ALP), Aminopeptidase N (APN) and Dipeptidyl Peptidase IV (DPP IV) activity were assayed as known markers of intestinal cell differentiation. The measure of TEER and the apparent permeability of Lucifer Yellow allows to monitor the integrity of the tight junctions and the permeability of the cell layer formed. At T0 a classical monolayer is present, with a mixed population of immature absorptive elements and secretive cells. At T6 and T14, cells are progressively organized in a multilayer with a parallel growth of microvilli. At T6, co-culture demonstrates good properties of permeability and barrier components, such as mucus, representing an appropriate model for absorption study. At T14, the brush border is even more developed respect to T6 and, together with the increase of the specific activity of ALP, APN, and DPP IV, indicate co-culture as a good model for digestion study. The advantage of this co-culture described is the use of the whole cell population without particular inducers of subclones and growth supports. In conclusion, the morphological and biochemical features of co-cultured parental cells change with time, strongly supporting i) an active interaction between the two parental cell lines and ii) the versatility of this model, with more than one prevalent cell type depending on the post confluent stage

Morphological analysis of the acute effects of cigarette smoke on normal human oral mucosa explants

Cigarette smoke is one of the five leading risk factors for mortality in the world and smoking related lung cancer is the main cause of deaths from cancer in both sexes in the United States. This habit is particularly popular in Western low- and middle- income countries, with about one billion males and 250 million females smoking in the world. Human oral mucosa is the combustion chamber of cigarette, but scanty evidence is available about the early smoke effects. The present work aimed at evaluating from a morphological point of view whole smoke early effects on epithelial intercellular adhesion and keratinocyte terminal differentiation in a three-dimensional model of human oral mucosa. Biopsies of keratinized oral mucosa of healthy non smoking women (n = 5) were collected. After culturing in a Transwell system, one fragment of each biopsy was exposed to the smoke of one single cigarette; the remnant represented the internal control. The distribution of epithelial differentiation markers (keratin-10, K10, and keratin-14, K14, for suprabasal and basal cells respectively), desmosomes (desmoglein-1, desmoglein-3), tight junctions (occludin), adherens junctions (E-cadherin, \u3b2-catenin), and apoptotic cells (p53, caspase-3) were evaluated by immunofluorescence. Quantitative analysis of K14 immunolabeling revealed an over expression in the suprabasal layers as early as 3 hours after smoke exposure, without impairment of the epithelial junctional apparatus and apoptosis induction. The present study showed that the early response of the normal human oral mucosa to acute exposure to the smoke of one single cigarette induced K14 expression in suprabasal oral keratinocytes, without impairment of the epithelial junctional apparatus and apoptosis induction, suggesting that the first significant response to cigarette smoke arise from the basal and suprabasal layers of the human oral epithelium. The novelty of the present work consists in the combination of human oral explants with a non hermetic smoke exposure system. The main improvement offered by this setting is the ability of reproducing the cyclic condition by which cigarette smoke normally comes in contact with the oral mucosa. Furthermore, it allows administering simultaneously both gaseous phase and particulate matter of cigarette smok